Role of the P2X7 nucleotide receptor in intestinal epithelial cell proliferation and differentiation

细胞外核苷酸对P2X配体门控离子通道的刺激与多种生理效应有关，包括细胞增殖和分化、蛋白质合成、细胞分泌和吸收的调节以及凋亡的调节。肠隐窝-绒毛轴是一个动态的自我更新系统，受不同的细胞和分子机制调控，参与细胞增殖、分化和凋亡。我们的假设是P2受体可能在这些生理过程的稳态中发挥重要作用。免疫组织化学研究显示P2X受体在隐窝-绒毛轴上的表达存在差异，提示不同的P2X受体在肠上皮细胞（IEC）成熟过程中发挥不同的作用。P2X7受体（P2X7R）是P2X家族的独特成员。它响应配体刺激形成一个孔，导致膜去极化和细胞凋亡。我们和其他人已经证明，短暂的P2X7R刺激会导致MAP激酶激活。根据这些数据，我们假设P2X7R不仅应该被视为一种“自杀受体”，而且还具有调节细胞成熟的受体。在此背景下，本研究计划将重点揭示肠上皮细胞（IEC）成熟过程中依赖于P2X7R活性的功能分子和细胞机制。首先，我们提出表征与IEC成熟相关的P2X7R信号转导途径。这将使用多种细胞和分子方法进行研究，包括干扰RNA (siRNA)， Western blotting和体外激酶测定。在第二个目标中，我们将确定P2X7R启动子调控元件和相关转录因子在成熟细胞中维持受体表达的重要作用。鉴定的转录因子的重要性将在体外通过siRNA和染色质免疫沉淀测定（ChIP）进行验证。本研究计划将确定调控P2X7R表达的分子和细胞机制及其在IEC分化中的假定作用。

Stimulation of P2X ligand-gated ion channels by extracellular nucleotides is linked to multiple physiological effects ranging from cellular proliferation and differentiation, protein synthesis, regulation of cell secretion and absorption as well as modulation of apoptosis. The intestinal crypt-villus axis is a dynamic self-renewal system that is regulated by different cellular and molecular mechanisms involved in cell proliferation, differentiation and apoptosis. Our hypothesis is that P2 receptors could play an important role in the homeostasis of these physiological process. Immunohistochemistry studies showed a differential pattern of P2X receptors expression along the crypt-villus axis, suggesting that different P2X receptors play different role in the maturation of intestinal epithelial cells (IEC). The P2X7 receptor (P2X7R) is a unique member of the P2X family. It forms a pore in response to ligand stimulation leading to membrane depolarization and apoptosis.   We and others have shown that transient P2X7R stimulation lead to MAP kinases activation. In lights of those data, we hypothese, that P2X7R should not only be viewed as a "suicidal receptor" but also has a receptor regulating cell maturation. In that context, this research program will focus to unravel the functional molecular and cellular mechanisms dependant on P2X7R activity during intestinal epithelial cells (IEC) maturation. First, we propose to characterize the P2X7R signal transduction pathways associated with IEC maturation.  This will be investigated with the use of diverse cellular and molecular approaches, including interference RNA (siRNA), Western blotting and in vitro kinase assays. In a second aim, we will identify the P2X7R promoter regulatory elements and associated transcription factors important in sustaining receptor expression in maturating cells. Validation of the importance of the identified transcription factors will be assayed in vitro by siRNA and by chromatin immunoprecipitation assays (ChIP). This research program will define the molecular and cellular mechanisms governing the regulation of P2X7R expression and its putative role in IEC differentiation.